PROJECT SUMMARY The objective of this study is to engineer an artificial T cell-stimulating matrix (aTM) that presents antigen-specific and cell-specific biochemical and biophysical cues to control phenotype and improve functional profiles of T cells in a biomimetic context that captures key biochemical and biophysical features of the lymph node (LN). LN provides the critical microenvironment that orchestrates the presentation format and dynamics of the antigen-specific signals in a precise and controlled manner that leads to T cell activation, expansion, and maturation. Currently available T cell stimulating matrices while carrying the antigen-presentation complexes and co-stimulating cues, however, lacks adequate supporting cues inherent to the T cell stimulating microenvironment in the LN. We will design and characterize an aTM that integrates the three key T-cell stimulating signals: antigen-specific (Signal 1), co-stimulatory (Signal 2), and cytokines (Signal 3), together with extracellular matrix (ECM) molecules and tunable biophysical properties inspired by the properties of both antigen-presenting cells (APCs) and the LN. We will test the hypothesis that co-presenting the APC signal cues in such a biomimetic context during T cell stimulation will result in functional T cells with controlled phenotypic profiles and potency. and mechanism revealing how all signaling cues synergize to active and polarize T cells. In Specific Aim 1, we will determine key matrix properties of the aTM that mediate robust CD8+ T cell activation using aTMs prepared from hyaluronic acid (HA) hydrogel and HA-nanofiber composite (NHC) matrix. In Specific Aim 2, we will define optimal signaling cues and their presentation configuration that influence CD4+ T cell activation and polarization on aTM; and demonstrate persistence and functions of aTM-stimulated and adoptively transferred antigen-specific CD4+ T cells. In Specific Aim 3, we will use aTM microparticles to co-stimulate CD8+ and CD4+ T cells and evaluate therapeutic benefits of a combination treatment with CD4+ and CD8+ T cells in a cancer immunotherapy mouse model. If successful, this study will result in the first set of engineered aTM that delivers robust stimulation of antigen-specific CD8+ and CD4+ T cells that promise improved therapeutic outcomes; and further enrich our understandings of design principles and mechanism of signaling cues in stimulation, polarization, and activation of T cells.